The present invention relates to a novel coronavirus (referred to herein as SARS-CoV) and to SARS-CoV vaccine compositions and methods of treating or preventing SARS-CoV infection and disease in mammals. SARS-CoV was discovered in March of 2003, in association with Severe Acute Respiratory Syndrome (SARS), a newly emerging infectious disease of global importance.
The recognition of SARS has led to activation of a global response network, with resultant travel restrictions, major quarantine, and closure of health care facilities. As of May 14, 2003, 7628 cases and 587 deaths from SARS have been reported from 29 countries. Initial reports of an atypical pneumonia began to surface in November of 2002 from the Guangdong province of China. This early outbreak reportedly involved 305 people, many of whom were healthcare workers. On Feb. 21, 2003, a healthcare worker from Guangdong traveled to Hong Kong, where his pre-existing cold symptoms escalated and he was hospitalized for acute respiratory distress. From Hong Kong, the illness spread rapidly throughout Southeast Asia and to Canada from this one index case. Seven individuals can be linked to the index case through a stay on the ninth floor of the hotel he occupied during his first night in Hong Kong. Infected persons from three hospitals in the Hong Kong metropolitan area are traceable to this index case as well. The primary mode of transmission has been either person-to-person contact or droplet transmission. Two notable exceptions to this are the hotel in Hong Kong, where direct human contact cannot be established for all those infected, and the Amoy Garden apartment buildings where more than 221 residents have been infected. In the outbreak at the Amoy Garden apartments, an unknown environmental factor is suspected of playing a role in transmission.
The incubation period ranges on average between two and seven days. Onset of symptoms begins with a high fever associated with chills and rigors. Additional symptoms at onset may include headache, malaise, myalgia, mild respiratory symptoms and more rarely common cold symptoms such as sore throat and runny nose. After this initial three to seven day period, additional lower respiratory symptoms appear including dry, non-productive cough and dyspnea. Initial chest x-rays reveal small, unilateral, patchy shadowings that progress quickly to bilateral, diffuse infiltrates. Preliminary. Outbreak news: severe acute respiratory syndrome (SARS). Wkly. Epidemiol. Rec., 2003: 81-88 (2003). The median duration of symptoms in a small epidemiologic study was 25.5 days. Tsang, K.W., et al. A cluster of cases of severe acute respiratory syndrome in Hong Kong, N. Engl. J. Med. (2003). The severity of illness can range widely from a mild illness to acute respiratory failure resulting in death. Patients with a significant co-morbidity, such as diabetes, or who are older, are more likely to suffer from a severe form of the disease. Questions remain as to why some patients become infected, while others who have intimate contact with infected individuals are spared. It does appear that patients are very contagious at the onset of symptoms. Studies from hospitals in Hong Kong and Hanoi have shown attack rates >56% among healthcare workers caring for SARS patients. It is unclear at this time whether individuals are contagious during the incubation phase.
Important Features of Coronaviruses
Coronaviruses are large, enveloped, positive-stranded RNA viruses, and they are known to elicit coincident diseases in animals and humans. Mature human coronavirus (HCoV) virions are approximately 100 nm-diameter enveloped particles exposing prominent spike (S), hemagglutinin-esterase (HE) (in some types of coronaviruses), envelope (E) and membrane (M) glycoproteins. Each particle contains an approximately 30 kilobase (kB) RNA genome complexed with an approximately 60 kilodalton (kD) nucleoprotein (N). Fields, B.N. VIROLOGY New York: Lippincott, Williams & Wilkins, (Fields, B.N., ed. 2001). All of the above references are herein incorporated by reference in their entireties.
The S proteins of HCoV's have two large domains, the variable S1 domain responsible for host cell binding, Breslin, J.J. et al. J Virol. 77: 4435-8 (2003), and the S2 domain containing a heptad coiled-coiled structure reminiscent of those involved in fusion in HIV and influenza. Yoo, D.W. et al. Virology 183: 91-8 (1991). The HCoV-229E, group I S protein appears to bind to the human aminopeptidase N glycoprotein, Yeager, C.L., et al. Nature 357: 420-2 (1992); Bonavia, A. et al. J. Virol. 77: 2530-8 (2003), whereas the HCoV-OC43 strain (HCoV-OC43, group II) may bind via sialic acid moieties. Vlasak, R. et al. Proc. Natl. Acad. Sci. USA 85:4526-9 (1988). The genetic variability between strains of coronavirus has not been thoroughly evaluated, although only minor variability has been observed in the S protein in the small number of strains sequenced. Hays, J.P. and Myint, S.H. J. Virol. Methods 75: 179-93 (1998); Kunkel, F. and Herrler, G. Arch. Virol. 141: 1123-31 (1996). Most coronaviruses are not only species specific, but also somewhat tissue tropic. This tropism is mostly related to changes in the S protein. Sanchez, C.M. et al. J Virol. 73: 7607-18 (1999). Examples of such coronavirus tropism changes are the in vitro demonstration that tropism can be experimentally manipulated by genetically replacing a feline S protein with a mouse S protein, and the natural emergence of the porcine respiratory coronavirus (PRCoV) from the transmissible gastroenteritis virus of swine (TGEV) strain merely through a deletion of a region in the S protein. Haijema, B.J. et al. J. Virol. 77:4528-38 (2003); Page, K.W. et al. J. Gen. Virol. 72:579-87 (1991); Britton, P. et al. Virus Res. 21:181-98 (1991). All of the above references are herein incorporated by reference in their entireties.
The recently discovered novel coronavirus, SARS-CoV, appears to be a new member of the order Nidovirales. Concerted efforts by many laboratories worldwide has led to the rapid sequencing of various strains of SARS-CoV, including CUKH-Su10 (GenBank Accession No. AY282752), TOR2 (GenBank Accession No. AY274119 and NC.sub.-004781), BJ01 (GenBank Accession No. AY278488), CUHK-W1 (GenBank Accession No. AY278554), Urbani (GenBank Accession No. AY278741) and HKU-39849 (GenBank Accession No. AY278491). The Urbani strain of SARS-CoV, sequenced by the Centers for Disease Control in Atlanta, Ga., is a 29,727-nucleotide, polyadenylated RNA with a genomic organization that is typical of coronaviruses: 5′-replicase, spike (S), envelope (E), membrane (M)-3′. Rota et al., Science 300:1394-1399 (2003), (hereinafter “Rota et al.”). In addition, there are short untranslated regions at both termini, and open reading frames (ORFs) encoding non-structural proteins located between S and E, between M and N, or downstream of N. Rota et al. The hemagglutinin-esterase (HE) gene found in group 2 and some group 3 coronaviruses was not found in SARS-CoV. Rota et al. Sequencing of the Tor2 SARS-CoV strain by a collaboration of researchers in British Columbia, Canada, yielded a genomic sequence that differed from the Urbani SARS-CoV strain by eight nucleotide bases. Marra et al., Science 300:1399-1404 (2003), (hereinafter “Marra et al.”). A comparison of the HKU-39849 and CUHK-W1 SARS-CoV strains also differed from the Urbani sequence by 10 or fewer nucleotide bases. Rota et al. All of the above references are herein incorporated by reference in their entireties.
Phylogenetic analyses indicate that, based on the genetic distance between SARS-CoV and other known coronaviruses in all of their genetic regions, no large region of the SARS-CoV genome was derived from other known viruses, and that SARS forms a distinct group within the genus Cornavirus. Rota et al.; Marra et al. The analyses also showed greater sequence conservation among enzymatic proteins of SARS-CoV than among the S, N, M, and E structural proteins; and, while there were regions of amino acid conservation within each protein as between SARS-CoV and other coronaviruses, the overall similarity was low. Rota et al. All of the above references are herein incorporated by reference in their entireties.
A virus, almost identical to the human SARS-CoV virus, has been isolated from rare Chinese masked palm civet cats. This virus is believed to be identical to human SARS-CoV except for a 29 nucleotide deletion in the region encoding the N protein of the virus. Walgate, R. “Human SARS virus not identical to civet virus” The Scientist, May 27, 2003, incorporated herein by reference in its entirety.
Coronavirus Vaccine Candidates
Because SARS-CoV was so recently discovered, there are no vaccines against the virus. The approach to vaccine development can, however, be partially guided by the results of past studies in animals, of which three diseases have received the greatest attention. These are transmissible gastroenteritis virus (TGEV) in swine, feline infectious peritonitis virus (FIPV), and avian infectious bronchitis virus (IBV). Of note, none of the vaccines, most of which have been attenuated vaccines, have proven to be highly efficacious except for inactivated IBV. Enjuanes, L. et al., Adv. Exp. Med. Biol. 380: 197-211 (1995). The FIPV vaccine is a live attenuated virus that has provided minimal efficacy in field trials, and the TGEV vaccine has also been problematic. Scott, F. W., Adv. Vet. Med. 41:347-58 (1999); Sestak, K. et al., Vet. Immunol. Immunopathol. 70:203-21 (1999). All of the above references are herein incorporated by reference in their entireties.
In the TGEV model, the major focus has been on neutralizing antibody directed at the S glycoprotein. Sestak, K. et al., Vet. Immunol. Immunopathol. 70: 203-21 (1999); Tuboly, T. et al. Vaccine 18: 2023-8 (2000); Shoup, D.I. et al. Am. J. Vet. Res. 58: 242-50 (1997). Protection has also been associated with antibodies in IBV and bovine coronavirus. Mondal, S. P. et al. Avian. Dis. 45:1054-9 (2001); Yoo, D.W. et al. Virology 180: 395-9 (1991). In fact, in most of the animal models, control of coronavirus infection can be due to antibodies reactive to the N-terminal region of the S protein. Gallagher, T.M. and Buchmeier, M.J. Virology 279: 371-4 (2001); Tuboly, T. et al. Arch. Virol. 137: 55-67 (1994). In one study of respiratory bovine coronavirus, antibody appearance to the S and N proteins was correlated with recovery. Lin, X.Q. et al. Arch. Virol. 145: 2335-49 (2000); Passive transfer studies have also been successful and demonstrated the value of humoral immune responses. Enjuanes, L. et al., Adv. Exp. Med. Biol. 380: 197-211 (1995); Spaan, W.J. Adv. Exp. Med. Biol. 276: 201-3 (1990). All of the above references are herein incorporated by reference in their entireties.
Cell-mediated immune responses have been most clearly detected in coronaviruses against the S, M and N proteins. Spencer, J.S. et al. Adv. Exp. Med. Biol. 380: 121-9 (1995); Collisson, E.W. et al. Dev. Comp. Immunol. 24: 187-200 (2000); Stohlman, S.A. et al. Virology 189: 217-24 (1992). In one study, the use of a DNA vaccine encoding the carboxyl terminus of the N gene of IBV, which induced cytotoxic T cell (CTL) activity, was able to decrease virus titers by 7 logs in target organs. Seo, S.H. et al. J. Virol. 71: 7889-94 (1997). Some protection was also noted in a DNA vaccine encoding the N protein in the Mouse Hepatitis Virus (MHV) model. Hayashi, M. et al. Adv. Exp. Med. Biol. 440:693-9 (1998). There is also some evidence that CTL may be involved in the control of MHV, and prevent the development of persistent infection and neuropathology. Pewe, L. and Perlman, S. Virology 255: 106-16 (1999); Pewe, L. et al. J. Virol. 71: 7640-7 (1997). All of the above references are herein incorporated by reference in their entireties.
A large number of coronavirus challenge studies have been conducted in humans by Tyrrell and colleagues, in which the subjects were inoculated intranasally and followed. Callow, K.A. et al. Epidemiol. Infect. 105: 435-46 (1990); Bende, M. et al. Acta Otolaryngol. 107: 262-9 (1989). Such challenge studies will clearly be impossible for the much more serious SARS-CoV virus. The presence of antibodies to the challenge strain did not prevent infection or disease, even in the face of rising neutralizing antibody titers. However, a second infection with similar strains led to decreased symptoms, revealing persistence of immunity against homologous challenge. Reed, S.E. J. Med. Virol. 13: 179-92 (1984). Also, the 2-4 year cyclical nature of the disease points to some persistence of immune response over time. Reed, S.E. J. Med. Virol. 13: 179-92 (1984); Hendley, J.O. et al. Am. Rev. Respir. Dis. 105: 805-11 (1972); Evans, A.S. and Kaslow, R.A. VIRAL INFECTIONS OF HUMANS. 4th ed. New York and London: Plenum Medical Book Company, (Evans, A.S. and Kaslow, R.A., eds., 1997). All of the above references are herein incorporated by reference in their entireties.
Heterologous “prime boost” strategies have been effective for enhancing immune responses and protection against numerous pathogens. Schneider et al., Immunol. Rev. 170:29-38 (1999); Robinson, H.L., Nat. Rev. Immunol. 2:239-50 (2002); Gonzalo, R.M. et al., Vaccine 20:1226-31 (2002); Tanghe, A., Infect. Immun. 69: 3041-7 (2001). Providing antigen in different forms in the prime and the boost injections appears to maximize the immune response to the antigen. DNA vaccine priming followed by boosting with protein in adjuvant or by viral vector delivery of DNA encoding antigen appears to be the most effective way of improving antigen specific antibody and CD4+ T-cell responses or CD8+ T-cell responses respectively. Shiver J.W. et al., Nature 415: 331-5 (2002); Gilbert, S.C. et al., Vaccine 20:1039-45 (2002); Billaut-Mulot, O. et al., Vaccine 19:95-102 (2000); Sin, J.I. et al., DNA Cell Biol. 18:771-9 (1999). Recent data from monkey vaccination studies suggests that adding CRL1005 poloxamer to DNA encoding the HIV gag antigen enhances T-cell responses when monkeys are vaccinated with an HIV gag DNA prime followed by a boost with an adenoviral vector expressing HIV gag (Ad5-gag). The cellular immune responses for a DNA/poloxamer prime followed by an Ad5-gag boost were greater than the responses induced with a DNA (without poloxamer) prime followed by Ad5-gag boost or for Ad5-gag only. Shiver, J.W. et al. Nature 415:331-5 (2002). U.S. Patent Appl. Publication No. US 2002/0165172 A1describes simultaneous administration of a vector construct encoding an immunogenic portion of an antigen and a protein comprising the said immunogenic portion of an antigen such that an immune response is generated. The document is limited to hepatitis B antigens and HIV antigens. Moreover, U.S. Pat. No. 6,500,432 is directed to methods of enhancing an immune response of nucleic acid vaccination by simultaneous administration of a polynucleotide and polypeptide of interest. According to the patent, simultaneous administration means administration of the polynucleotide and the polypeptide during the same immune response, preferably within 0-10 or 3-7 days of each other. The antigens contemplated by the patent include, among others, those of Hepatitis (all forms), HSV, HIV, CMV, EBV, RSV, VZV, HPV, polio, influenza, parasites (e.g., from the genus Plasmodium), pathogenic bacteria (including but not limited to M tuberculosis, M leprae, Chlamydia, Shigella, B. burgdorferi, enterotoxigenic E. coli, S. typhosa, H. pylori, V. cholerae, B. pertussis, etc.). All of the above references are herein incorporated by reference in their entireties.